EP1527202A1 - Couches d'identification constituees d'hydrogel a base de polyacrylamide destinees a des capteurs biologiques - Google Patents

Couches d'identification constituees d'hydrogel a base de polyacrylamide destinees a des capteurs biologiques

Info

Publication number
EP1527202A1
EP1527202A1 EP03790648A EP03790648A EP1527202A1 EP 1527202 A1 EP1527202 A1 EP 1527202A1 EP 03790648 A EP03790648 A EP 03790648A EP 03790648 A EP03790648 A EP 03790648A EP 1527202 A1 EP1527202 A1 EP 1527202A1
Authority
EP
European Patent Office
Prior art keywords
immobilization layer
layer according
hydrophilic
polyacrylamide
hydrophilic immobilization
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03790648A
Other languages
German (de)
English (en)
Inventor
Hans-Dieter Feucht
Walter Gumbrecht
Manfred Stanzel
Heinrich Zeininger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens AG
Original Assignee
Siemens AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Siemens AG filed Critical Siemens AG
Publication of EP1527202A1 publication Critical patent/EP1527202A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/002—Electrode membranes

Definitions

  • the present invention relates to an immobilization layer for biosensors and their use for the production of biosensory detection layers, in particular for the production of so-called DNA chips.
  • biosensors are increasingly used in which a biological detection system is linked to a physical transducer.
  • Recognition systems are understood to be biological recognition molecules, such as antibodies, enzymes, nucleic acids and the like, which are bound to a support (transducer) via a so-called immobilization layer.
  • Transducer a support
  • immobilization layer a support
  • Mainly calorimetric, piezoelectric, optical and electrochemical principles are used as transducers.
  • the detection systems or originally the immobilization layers, are usually immobilized in approximately two-dimensional layers on the transducer systems.
  • the recognition molecules can be immobilized by 5 covalent bonds, by affinity interaction, but also by hydrophilic / hydrophobic interactions. For reasons of stability, covalent bonds are preferred, but the formation of stable complexes, such as biotin / avidin, is also successfully used.
  • a good overview of the structure of almost two-dimensional biological recognition layers is given by I. Willner, E. Katz: "Redox protein layers on conductive supports - systems for bioelectronic applications" in Angew. Chem. 2000, 112, pp. 1230-69.
  • the biological function carriers ie the recognition molecules
  • the biological function carriers are often replaced by alkoxysilanes, which are known as contain linker groups, but are also immobilized with the aid of cyanuric chloride or carbodiimide.
  • recognition molecules labeled with tiolalkyl are used, which are immobilized on the transducer surface via sulfur-gold bonds in the form of so-called self-assembly layers.
  • WO 00/43539 describes the construction of a three-dimensional DNA recognition layer by immobilizing the DNA capture probes in the form of polymer brushes.
  • Timofeev et al. describes a chemically modified, radically crosslinked polyacrylamide which can be used, for example, for immobilizing capture oligos (EN Timofeev et al., Regioselective Immobilization of Short Oligonucleotides to Acrylic Copolymer Gels, Nucleic Acids Research, 1966, Vol. 24, 16, 3142-3148).
  • amino or aldehyde groups are used as coupling groups in the hydrogel.
  • Aldehyde- or amino-functionalized scavenger oligos can be covalently immobilized on these coupling groups under reductive reaction conditions.
  • the object of the present invention is to produce a hydrophilic immobilization layer for biosensor applications based on a hydrogel and to use such immobilization layers to generate recognition layers by covalently coupling biological recognition molecules.
  • the present invention solves this problem by using free-radically crosslinked or photo-structured hydrogels as the immobilization layer.
  • hydrogels are described in the applicant's German patent applications "Radically crosslinkable composition for producing a hydrogel layer” or "Photo-structurable composition for producing a hydrogel layer” (file number not yet known).
  • the present invention accordingly relates to a hydrophilic immobilization layer for biosensors made from a free-radically crosslinked hydrogel based on polyacrylamide, the starting composition comprising acrylamide, crosslinking agents, radical initiators, at least one comonomer with reactive linker groups and optionally plasticizers and other additives.
  • the subject of the present compound is also a hydrophilic immobilization layer made of a photostructured hydrogel based on polyacrylamide, the starting composition being acrylamide, crosslinking agent, photoinitiators, at least one film former, at least one comonomer with reactive linker groups and optionally plasticizers and other additives.
  • the systems according to the invention allow the construction of sensor arrays with biological recognition molecules in a three-dimensional matrix with a high integration density.
  • compositions which ensure the miscibility of the monomers involved and the initiators.
  • Commercial additives can be used to reduce the surface tension.
  • a water-swellable hydrogel is obtained, into which biological or chemical recognition molecules for analytical or diagnostic applications are coupled in using the linker groups while maintaining their functionality can.
  • the present invention accordingly also relates to the use of the immobilization layers for the production of biosensory recognition layers by (covalent) coupling or immobilization of chemical or biological recognition molecules, the recognition molecules preferably being scavenger oligonucleotides.
  • the starting composition for generating the hydrogel layer (immobilization layer) can be applied to the suitable carrier using all modern coating technologies. However, spin coating and dispensing are preferably used.
  • the properties of the hydrogel layer to be produced with regard to hydrophilicity, crosslinking density, swellability, etc. can be varied widely by the type of starting components used, their relationship to one another and ultimately the type of layer formation.
  • the hydrogel matrix can be adapted to the biological recognition molecules used, in particular with regard to the crosslinking density.
  • the crosslinking density will be controlled by the type and concentration of the crosslinking molecules used, such as acrylic and / or methacrylic compounds, in particular methylenebis (meth) acrylamide and / or dimethacrylic acid esters, such as tetraethylene glycol dimethacrylate.
  • the hydrogel mixture can also be adapted to the coating method preferred for the specific application.
  • a polymeric film former such as polyvinylpyrrolidone, polyacrylamide and / or polyhydroxymethacrylate
  • high-boiling solvents such as.
  • ethylene glycol can be used for the hydrogel mixture, which do not evaporate completely during spin coating and thus as a plasticizer in the
  • the residual solvent content can then be further reduced in a targeted manner by means of a prebake step before crosslinking, and thus the polymerization yield or the resulting layer thickness can be controlled. If necessary, additional plasticizer systems, such as di- and / or triethylene glycol, can be added.
  • the hydrogel mixture in solution is applied in drops, depending on the transducer dimensions, in sizes from a few microliters to one nanoliter.
  • the dispensing are high-boiling solvents, which have a long enough life for the drop have at the tip of the dispensing cannula used. This makes the dosing and settling of the drop reproducible.
  • the boiling point of the solvent must not be too high to allow the solvent to evaporate sufficiently quickly from the settled drop.
  • a tempering step to control the residual solvent content may be required. According to the invention, preference is given to using dimethylformamide and / or ethylene glycol for dispensing the hydrogel mixture.
  • the hydrogel mixture can be applied in layer or spot form on transducer or carrier surfaces made of metal, glass, silicon, silicon dioxide, silicon nitride or plastic.
  • Surfaces with topography which consist of different materials, such as.
  • B. Interdigital electrode arrays are coated on silicon nitride as passivation.
  • the coating of surfaces also includes the coating of inner surfaces of microchannels or nanotubes.
  • the surfaces to be coated are optionally coated with an adhesion promoter.
  • the hydrogel layer is polymerized and crosslinked by thermal or UV initiation.
  • the hydrogel layer can also be structured by contact or proximity exposure through a mask.
  • the hydrogel layer works like a negative resist. Polymerization and crosslinking take place in the irradiated area. There is no reaction in the darkened areas.
  • the hydrogel mixture found here is detached from the substrate in a development step. Auxiliary components such as polymeric film formers or plasticizers can be removed from the crosslinked hydrogel layer by extraction. Under certain circumstances, this step can take place at the same time as the actual equipment step.
  • the bioglogical or chemical detection systems are preferably made from aqueous solution, from aqueous buffer solution or applied to the immobilization layer from mixtures of polar solvents with water. It is applied by dripping on or spotting on / dispensing.
  • the solution with the biological or chemical recognition molecules can also be brought to the crosslinked hydrogel layer by transport through the fluidic system itself.
  • Cross-linked hydrogel spots which are surrounded by a protective ring, are advantageously used for the precise loading of measuring spots.
  • a tempering step may be required for the covalent coupling of the biological or chemical recognition molecules, which are provided with a coupling group that matches the linker group present in the crosslinked hydrogel.
  • a inoalkyl groups are particularly suitable for coupling to the linker groups epoxy and maleic anhydride.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une couche d'immobilisation hydrophile destinée à des capteurs biologiques, constituée d'un hydrogel à base de polyacrylamide pouvant être réticulé de façon radicalaire, la composition initiale contenant acrylamide, des agents réticulants, un ou plusieurs initiateurs de radicaux, au moins un comonomère comportant des groupes liants réactifs, et éventuellement des agents plastifiants. Ladite couche d'immobilisation hydrophile peut également être constituée d'un hydrogel photostructuré à base de polyacrylamide, la composition initiale contenant alors acrylamide, des agents réticulants, des photoinitiateurs, au moins un agent filmogène, au moins un comonomère comportant des groupes liants réactifs, et éventuellement des agents plastifiants.
EP03790648A 2002-08-08 2003-07-23 Couches d'identification constituees d'hydrogel a base de polyacrylamide destinees a des capteurs biologiques Withdrawn EP1527202A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10236459 2002-08-08
DE10236459 2002-08-08
PCT/DE2003/002483 WO2004020659A1 (fr) 2002-08-08 2003-07-23 Couches d'identification constituees d'hydrogel a base de polyacrylamide destinees a des capteurs biologiques

Publications (1)

Publication Number Publication Date
EP1527202A1 true EP1527202A1 (fr) 2005-05-04

Family

ID=31968951

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03790648A Withdrawn EP1527202A1 (fr) 2002-08-08 2003-07-23 Couches d'identification constituees d'hydrogel a base de polyacrylamide destinees a des capteurs biologiques

Country Status (4)

Country Link
US (1) US20060111517A1 (fr)
EP (1) EP1527202A1 (fr)
JP (1) JP2005534942A (fr)
WO (1) WO2004020659A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060257560A1 (en) * 2004-12-30 2006-11-16 Affymetrix, Inc. Polymer surfaces for insitu synthesis of polymer arrays
US20060174385A1 (en) * 2005-02-02 2006-08-03 Lewis Gruber Method and apparatus for detecting targets
JP4689475B2 (ja) * 2006-01-11 2011-05-25 ニプロ株式会社 核酸固定用成形体および核酸固定化方法
WO2008140562A2 (fr) 2006-11-03 2008-11-20 Trustees Of Tufts College Dispositifs optiques et électro-optiques à biopolymère électroactif et leur procédé de fabrication
JP2010509645A (ja) 2006-11-03 2010-03-25 トラスティーズ オブ タフツ カレッジ ナノパターンが形成されたバイオポリマー光学デバイスおよびその製造方法
EP2101975A2 (fr) * 2006-11-03 2009-09-23 Trustees of Tufts College Capteur de biopolymère et procédé de fabrication de celui-ci
WO2008118211A2 (fr) 2006-11-03 2008-10-02 Trustees Of Tufts College Cristaux photoniques biopolymères et procédés de fabrication de ceux-ci
US11697793B2 (en) * 2018-10-10 2023-07-11 Washington University Compositions and methods of making and using protein-functionalized hydrogels

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU664913B2 (en) * 1992-08-13 1995-12-07 Implico B.V. A hydrogel composition and methods of making it
US5372766A (en) * 1994-03-31 1994-12-13 The Procter & Gamble Company Flexible, porous, absorbent, polymeric macrostructures and methods of making the same
US5596038A (en) * 1994-05-16 1997-01-21 Physiometrix, Inc. Hydrogel having a silicon-based crosslinker for biosensors and electrodes
US5932711A (en) * 1997-03-05 1999-08-03 Mosaic Technologies, Inc. Nucleic acid-containing polymerizable complex
US6391937B1 (en) * 1998-11-25 2002-05-21 Motorola, Inc. Polyacrylamide hydrogels and hydrogel arrays made from polyacrylamide reactive prepolymers
EP1144677B1 (fr) * 1999-01-25 2006-07-26 Micronas Holding GmbH Immobilisation de molecules sur des surfaces par des brosses polymeres
US6372813B1 (en) * 1999-06-25 2002-04-16 Motorola Methods and compositions for attachment of biomolecules to solid supports, hydrogels, and hydrogel arrays

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004020659A1 *

Also Published As

Publication number Publication date
WO2004020659A1 (fr) 2004-03-11
US20060111517A1 (en) 2006-05-25
JP2005534942A (ja) 2005-11-17

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